Saccharide compositions, methods and apparatus for their synthesis

ABSTRACT

A method for preparing saccharide compositions is disclosed. The method is reiterative and comprises the following three steps.  
     (i) A glycosyltransferase capable of transferring a preselected saccharide unit to an acceptor moiety is isolated by contacting the acceptor moiety with a mixture suspected of containing the glycosyltransferase under conditions effective to bind the acceptor moiety and the glycosyltransferase and thereby isolate the glycosyltransferase. The acceptor moiety is a protein, a glycoprotein, a lipid, a glycolipid, or a carbohydrate.  
     (ii) The isolated glycosyltransferase is then used to catalyze the bond between the acceptor moiety and the preselected saccharide unit.  
     (iii) Steps (i) and (ii) are repeated a plurality of times with the intermediate product obtained in the first iteration of the method being used as the acceptor moiety of the second iteration.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 07/509,560, filed Apr. 16, 1990, and relates to subject matterdisclosed in copending U.S. patent application Ser. No. 07/241,012,filed Sep. 2, 1988, entitled “Carbohydrates and Carbohydrate Complexesfor Therapeutic and Preventative Treatment of Mammals”. Ser. No.07/241,012 is incorporated herein by reference

GOVERNMENT SUPPORT

[0002] Portions of this invention were supported by National ScienceFoundation Grant DCB8817883.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates to saccharide compositions such as, forexample, oligosaccharides, polysaccharides, glycolipids, andglycoproteins. More specifically, this invention relates to processesfor preparing these and other saccharide compositions by enzymatictechniques.

[0005] 2. Discussion of the Background

[0006] The term “carbohydrate” embraces a wide variety of chemicalcompounds having the general formula (CH₂0)n, such as monosaccharides,disaccharides, oligosaccharides and polysaccharides. Oligosaccharidesare chains composed of saccharide units, which are alternatively knownas sugars. These saccharide units can be arranged in any order and thelinkage between two saccharide units can occur in any of approximatelyten different ways. As a result, the number of different possiblestereoisomeric oligosaccharide chains is enormous.

[0007] of all the biological polymer families, oligosaccharides andpolysaccharides have been the least well studied, due in considerablepart to the difficulty of sequencing and synthesizing their oftencomplex sugar chains. Although the syntheses of oligonucleotides andpolypeptides are well developed, there is currently no generallyapplicable synthetic technique for synthesizing oligosaccharides.

[0008] Numerous classical techniques for the synthesis of carbohydrateshave been developed, but these techniques suffer the difficulty ofrequiring selective protection and deprotection. Organic synthesis ofoligosaccharides is further hampered by the lability of many glycosidicbonds, difficulties in achieving regioselective sugar coupling, andgenerally low synthetic yields. These difficulties, together with thedifficulties of isolating and purifying carbohydrates and of analyzingtheir structures, has made this area of chemistry a most demanding one.

[0009] Much research effort has been devoted to carbohydrates andmolecules comprising carbohydrate fragments, such as glycolipids andglycoproteins. Research interest in such moieties has been largely dueto the recognition that interactions between proteins and carbohydratesare involved in a wide array of biological recognition events, includingfertilization, molecular targeting, intercellular recognition, andviral, bacterial, and fungal pathogenesis. It is now widely appreciatedthat the oligosaccharide portions of glycoproteins and glycolipidsmediate recognition between cells and cells, between cells and ligands,between cells and the extracellular matrix, and between cells andpathogens.

[0010] These recognition phenomena can likely be inhibited byoligosaccharides having the same sugar sequence and stereochemistryfound on the active portion of a glycoprotein or glycolipid involved incell recognition. The oligosaccharides are believed to compete with theglycoproteins and glycolipids for binding sites on receptor proteins.For example, the disaccharide galactosyl β1-4 N-acetylglucosamine isbelieved to be one component of the glycoproteins which interact withreceptors in the plasma membrane of liver cell. Thus, to the extent thatthey compete with potentially harmful moieties for cellular bindingsites, oligosaccharides and other saccharide compositions have thepotential to open new horizons in pharmacology, diagnosis, andtherapeutics.

[0011] There has been relatively little effort to test oligosaccharidesas therapeutic agents for human or animal diseases, however, as methodsfor the synthesis of oligosaccharides have been unavailable as notedabove. Limited types of small oligosaccharides can be custom-synthesizedby organic chemical methods, but the cost for such compounds istypically very high. In addition, it is very difficult to synthesizeoligosaccharides stereospecifically and the addition of some sugars,such as sialic acid and fucose, has not been effectively accomplishedbecause of the extreme lability of their bonds. Improved, generallyapplicable methods for oligosaccharide synthesis are desired for theproduction of large amounts of widely varying oligosaccharides forpharmacology and therapeutics.

[0012] For certain applications, enzymes have been targeted for use inorganic synthesis as one alternative to more traditional techniques. Forexample, enzymes have been used as catalysts in organic synthesis; thevalue of synthetic enzymatic reactions in such areas as rateacceleration and stereoselectivity has been demonstrated. Additionally,techniques are now available for low cost production of some enzymes andfor alteration of their properties.

[0013] The use of enzymes as catalysts for the synthesis ofcarbohydrates has been proposed, but to date enzyme-based techniqueshave not been found which are useful for the general synthesis ofoligosaccharides and other complex carbohydrates in significant amounts.It has been recognized that a major limiting factor to the use ofenzymes as catalysts in carbohydrate synthesis is the very limitedcurrent availability of the broad range of enzymes required toaccomplish carbohydrate synthesis. See Toone et al, Tetrahedron Reports(1990) (45)17:5365-5422.

[0014] In mammalian systems, eight monosaccharides activated in the formof nucleoside mono- and diphosphate sugars provide the building blocksfor most oligosaccharides: UDP-Glc, UDP-GlcUA, UDP-GlcNAc, UDP-Gal,UDP-GalNAc, GGP-Man, GDP-Fuc and CMP-NeuAc. These are the intermediatesof the Leloir pathway. A much larger number of sugars (e.g., xylose,arabinose) and oligosaccharides are present in microorganisms andplants.

[0015] Two groups of enzymes are associated with the in vivo synthesisof oligosaccharides. The enzymes of the Leloir pathway is the largestgroup. These enzymes transfer sugars activated as sugar nucleosidephosphates to a growing oligosaccharide chain. Non-Leloir pathwayenzymes transfer carbohydrate units activated as sugar phosphates, butnot as sugar nucleoside phosphates.

[0016] Two strategies have been proposed for the enzyme-catalyzed invitro synthesis of oligosaccharides. See Toone et al, supra. The firststrategy proposes to use glycosyltransferases. The second proposes touse glycosidases or glycosyl hydrolases.

[0017] Glycosyltransferases catalyze the addition of activated sugars,in a stepwise fashion, to a protein or lipid or to the non-reducing endof a growing oligosaccharide. A very large number ofglycosyltransferases appear to be necessary to synthesize carbohydrates.Each NDP-sugar residue requires a distinct class of glycosyltransferasesand each of the more than one hundred glycosyltransferases identified todate appears to catalyze the formation of a unique glycidic linkage. Todate, the exact details of the specificity of the glycosyltransferasesare not known. It is not clear, for example, what sequence ofcarbohydrates is recognized by most of these enzymes.

[0018] Enzymes of the Leloir pathway have begun to find application tothe synthesis of oligosaccharides. Two elements are required for thesuccess of such an approach. The sugar nucleoside phosphate must beavailable at practical cost and the glycosyltransferase must beavailable. The first issue is resolved for most common NDP-sugars,including those important in mammalian biosynthesis. The problem in thistechnology however resides with the second issue. To date, only a verysmall number of glycosyltransferases are available. Access to thesetypes of enzymes has been the single limiting factor to this type ofcarbohydrate synthesis.

[0019] It has been reported that most glycosyltransferases are difficultto isolate, particularly from mammalian source. This is because theseproteins are present in low concentrations and are membrane-bound.Further, although a few glycosyltransferases have been immobilized,these enzymes have been reported to be unstable. To date only a verysmall number of glycosyltransferases are available from commercialsources, and these materials are expensive.

[0020] Much hope has therefore been put on future developments ingenetic engineering (i.e., cloning) of enzymes, particularly sinceseveral glycosyltransferases have already been cloned, includinggalacto-, fucosyl-, and sialyltransferases. It is hoped that futureadvances in cloning techniques will speed the cloning of otherglycosyltransferases and enhance their stability.

[0021] Accordingly, in light of their potential uses and the difficultyor impossibility to obtain them in sufficient quantities, there exists along-felt need for general synthetic methods for the production ofoligosaccharides, polysaccharides, glycoproteins, glycolipids, andsimilar species in an efficient, cost effective, stereospecific, andgenerally applicable manner.

SUMMARY OF THE INVENTION

[0022] It is an object of the present invention to provide saccharidecompositions, particularly oligosaccharides, polysaccharides andchemical moieties which comprise oligosaccharide units.

[0023] It is another object of this invention to provide a wide varietyof saccharide compositions, including those not found in nature.

[0024] It is a further object of this invention to provide saccharidecompositions useful in mitigating the effects of human or animaldiseases.

[0025] It is yet another object of this invention to provide improvedprocesses for preparing saccharide compositions.

[0026] It is a further object of this invention to provide enzymaticprocesses for preparing saccharide compositions.

[0027] It is still another object of this invention to provide processesfor obtaining enzymes useful in synthesizing saccharide compositions.

[0028] It is still another object of this invention to provide anapparatus useful for the synthesis of saccharide compositions inaccordance with the present invention.

[0029] These and other objects are achieved by the present invention,which provides enzymatic processes for preparing oligosaccharides,polysaccharides, glycolipids, glycoproteins, and other saccharidecompositions. These processes involve the enzyme-facilitated transfer ofa preselected saccharide unit from a donor moiety to an acceptor moiety.Saccharide compositions having a plurality of saccharide units arepreferably prepared by appending the saccharide units in stepwisefashion to acceptor moieties which are themselves saccharidecompositions prepared in accordance with this invention.

[0030] Accordingly, methods for preparing saccharide compositions areprovided comprising the steps of providing an acceptor moiety andcontacting the acceptor moiety with a glycosyltransferase. Theglycosyltransferase is prepared so as to be specific for the acceptormoiety and capable of transferring a saccharide unit to the acceptormoiety. This method of the present invention is performed a plurality oftimes such that the product of the first iteration becomes the acceptormoiety for a second iteration, and so forth.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIGS. 1, 2 and 3 illustrate apparatuses suitable for use in theglycosyltransferase catalyzed synthesis of saccharide composition inaccordance with the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] As employed herein, the term “saccharide composition” is intendedto include any chemical moiety having a saccharide unit within itsstructure. Sugars, carbohydrates, saccharides, monosaccharides,oligosaccharides, polysaccharides, glycoprotains, and glycolipidsprovide examples of saccharide compositions. Mixtures and solutionscomprising such moieties are also saccharide compositions.

[0033] Saccharide compositions are prepared according to this inventionby the enzyme facilitated transfer of saccharide units from donormoieties to acceptor moieties. It will be appreciated that such transferoccurs upon contacting the acceptor and donor moieties with aglycosyltransferase, and typically results in covalently bonding of theacceptor moiety and the saccharide unit stereoselectively, that is, inbut one- stereoisomeric form.

[0034] The saccharide compositions prepared in accordance with thisinvention are believed to find wide utility in diagnostics,therapeutics, and pharmacological applications. Once the sugar sequenceof a desired target saccharide composition has been determined byconventional methods, a retrosynthetic analysis is generally performedto determine an appropriate synthetic scheme for the saccharidecomposition. Such a synthetic scheme preferably identifies theparticular donor moieties, acceptor moieties, and glycosyltransferasesnecessary to yield the desired saccharide composition.

[0035] Instead of relying on future developments in genetic engineeringto provide the numerous qlycosyltransferases required for carbohydratesynthesis, the present invention relies on the very different approachas follows. In the synthesis of a saccharide composition in accordancewith the invention, a preselected saccharide unit is first enzymaticallyattached to an initial acceptor moiety, i.e., a protein, a glycoprotein,a lipid, a glycolipid, or a carbohydrate starting material. This isfollowed by enzymatically attaching preselected saccharide units to theproduct obtained in a stepwise fashion thereby forming the saccharidecomposition.

[0036] With the attachment of each preselected saccharide unit oneobtains an intermediate product. The present invention is based on theinventor's discovery that the starting material of the synthesis (i.e.,the protein, glycoprotein, lipid, glycolipid or carbohydrate) and eachintermediate product formed in the synthesis can be advantageously usedto obtain, for each corresponding step of the synthesis, aglycosyltransferase specific to catalyze the attachment of the nextintermediate product in the synthesis of the target saccharidecomposition.

[0037] Thus, in accordance with the invention, the glycosyltransferaseneeded for any given step is isolated with the intermediate product (theacceptor moiety) and used to attach to the acceptor moiety the nextsaccharide unit necessary for construction of the target carbohydratemolecule. In accordance with the present invention, this process isrepeated, with each iteration (time) yielding the particularglycosyltransferase required to attach the next saccharide unit onto thegrowing molecule being isolated, until the target carbohydrate moleculeis obtained.

[0038] Also provided by the invention are reaction conditions andco-reagents as may be necessary and sufficient to effect the covalentbonding of the saccharide unit to the acceptor moiety.

[0039] In accordance with preferred embodiments, the acceptor moiety maybe a protein, glycoprotein, lipid, glycolipid, or carbohydrate, such asa monosaccharide, disaccharide, oligosaccharide, or polysaccharide. Inaccordance with other preferred embodiments, the glycosyltransferase isattached to a solid support.

[0040] The present methods are capable of stereospecific attachment ofthe saccharide unit to the acceptor moiety. In general, it is preferredto employ saccharide nucleotides as donor moieties. Uridine, guanosine,and cytidine phosphate materials terminated by the saccharide units tobe donated preferably comprise the donor moieties.

[0041] The present invention thus also provides means for preparing aglycosyltransferase specific for a particular acceptor moiety andcapable of transferring a preselected saccharide unit to the acceptormoiety. Such methods comprise contacting the acceptor moiety with amixture suspected to contain a plurality of glycosyltransferases underconditions effective to bind the acceptor moiety and theglycosyltransferase specific for the acceptor moiety. The resulting,bound glycosyltransferase is subsequently isolated. It is preferred thatthe glycosyltransferase be sequenced and that the glycosyltransferase beproduced in enhanced quantities by genetic engineering techniques.

[0042] The mixture suspected to contain a glycosyltransferase ofinterest may be identified as follows. For the most common glycosidiclinkages, the glycosyltransferase activities have been described inpublications. This is largely true for compounds like milkoligosaccharides, or the carbohydrate moieties of typical (i.e.,prevalent) glycoproteins and glycolipids. For less well describedlinkages, one may first look to the tissue, organ, foodstuff organism,in which the linkage is found. Generally, if the linkage is found in aparticular source, the enzyme that made the linkage is also present inthe source.

[0043] If one is presented only with a saccharide structure, and not asource, one can then test examples of organisms that are likely tocontain such a saccharide structure using the most sensitive screeningassay available. For example, if the compound contained iduronic acid,N-acetylgalactosamine and N-acetylglucosamine, one would test vertebrateconnective tissue. If the target compound contain abequose, one wouldtest bacteria and plants for the presence of the appropriateglycosyltransferase.

[0044] Various assays for detecting glycosyltransferases which can beused in accordance with the invention have been published. The followingare illustrative. Furukawa et al, Biochem. J., (1985) 227:573-582describe a borate-impregnated paper electrophoresis assay and afluorescence assay (FIG. 6) developed by the inventor. Roth et al, ExP'lCell Research (1983) 143:217-225 describe application of the borateassay to glucuronyl transferases, previously assayed calorimetrically.Benau et al, J. Histochem. Cytochem. (1990) 38(1):23-30 describe ahistochemical assay based on the reduction, by NADH, of diazonium salts.

[0045] Once a source for the glycosyltransferase of interest has beenfound, the source is homogenized. The enzyme is purified from homogenateby affinity chromatography using the acceptor moiety as the affinityligand. That is, the homogenate is passed over a solid matrix havingimmobilized thereon the acceptor moiety under conditions which cause theglycosyltransferase to bind to the acceptor moiety. The solid supportmatrix having the glycosyltransferase bound thereto is then washed. Thisis followed by an elution step in which the glycosyltransferase isdesorbed from the solid support matrix and collected. As known, theabsorbed glycosyltransferase may be eluted, for example, by passing anaqueous salt (e.g. NaCl) solution over the solid support matrix.

[0046] In actual practice of the invention, the “enzyme” purified fromthe homogenate by affinity chromatography and which is used to attach apreselected saccharide unit onto the acceptor moiety comprises a mixtureof various glycosyltransferases which have been purified away from otherextraneous biological material present in the homogenate which includesenzymes which can interfere with the desired activity of the purifiedglycosyltransferases. Thus, the glycosyltransferases used in accordancewith the present invention are frequently a mixture of various“glycosyltransferase”. If desired, this material may be further purifiedwith a single purified glycosyltransferase being isolated and used inthe process of the present invention, but such further purification isgenerally not necessary.

[0047] In accordance with the present invention, an acceptor moiety isprovided which is capable of being covalently bound to a preselectedsaccharide unit. Representative acceptor moieties include proteins,glycoproteins, lipids, glycolipids and carbohydrates. It will beappreciated that acceptor moieties are preferred to the extent that theyare present as a structural component of a saccharide composition ofinterest. For example, in preparing a saccharide composition such asN-acetylneuraminyl β 2-3 galactosyl β 1-4 N-acetylglucosamine, preferredacceptor moieties would be N-acetylglucosamine and galactosyl β 1-4N-acetylglucosamine. It will likewise be appreciated that where anacceptor moiety is terminated by a saccharide unit, subsequentsaccharide units will typically be covalently bound to the nonreducingterminus of the terminal saccharide.

[0048] The saccharide unit to be transferred to an acceptor moiety isprovided by a donor moiety for the saccharide unit. A donor moietyaccording to this invention includes the saccharide unit to betransferred and is capable of providing that saccharide unit to theacceptor moiety when contacted by the acceptor moiety and theappropriate glycosyltransferase. Preferred donor moieties are saccharidenucleotides, such as saccharide-terminated uridine phosphates,saccharide-terminated quanosine phosphates, and saccharide-terminatedcytidine phosphates.

[0049] It will be appreciated that donor moieties are preferred to becapable of readily providing their component saccharide unit to anacceptor moiety when placed in contact therewith and with aglycosyltransferase. For example, uridine diphosphate galactose ispreferred for transferring galactose to N-acetylglucosamine, whilecytidine monophosphate N-acetylneuraminic acid is preferred fortransferring N-acetylneuraminic acid, a sialic acid, to galactosyl β 1-4N-acetylglucosamine.

[0050] Upon identification of acceptor moieties and donor moietiesnecessary for the preparation of a saccharide composition, aglycosyltransferase for each acceptor/donor pair should be prepared.Those skilled in the art will appreciate that a glycosyltransferase maybe broadly defined as an enzyme which facilitates the transfer of asaccharide unit from one chemical moiety (here defined as a donor) toanother (here defined as an acceptor) and which is namedphenomenologically according to the saccharide unit it transfers. Thus,galactosyltransferase transfers galactose, while fucosyltransferasetransfers fucose.

[0051] Glycosyltransferases according to this invention are those ableto effect the transfer of a predetermined saccharide unit to an acceptormoiety. Glycosyltransferases are preferably specific for an acceptormoiety or at least some significant, active, or exposed portion thereof.Specificity is manifested for a glycosyltransferase by its tendency tobind with a particularly sequenced portion of an acceptor moiety whenplaced in contact or close proximity therewith and to effect thetransfer of a particular saccharide unit to that acceptor moiety.

[0052] Currently, glycosyltransferases are available only from naturalsources and, as a result, are somewhat limited in number. It will beappreciated that known glycosyltransferases are only capable ofeffecting saccharide unit transfers which are highly specific, both interms of the chemical identity of the saccharide unit transferred andthe stereochemistry of its subsequent attachment to the acceptor moiety.For example, it is known that one N-acetylneuraminyltransferase caneffect the transfer of N-acetylneuraminic acid to an acceptor moietybearing only a galactose unit to produce a saccharide composition havingan α 2-3 linkage between the N-acetylneuraminic acid unit and thegalactose unit.

[0053] Thus, the invention permits construction of sugar linkages foundin nature. For example, the linkage of galactose α 1-2 toN-acetylneuraminic acid, which has not been found in nature, cannotpresently be effected. The methods disclosed herein are, however,applicable to any type of glycosyltransferase which may becomeavailable.

[0054] While the behavior of a number of glycosyltransferases is known,most glycosyltransferases are currently not fully characterized. Thepresent invention, however, provides methods by which allglycosyltransferases amenable to its practice may be identified andprepared. It has now been found that an acceptor moiety can be used asan affinity chromatographic tool to isolate enzymes that can be used totransfer particular saccharide units and, thus, synthesize otherglycosides.

[0055] In a preferred embodiment, an acceptor moiety is immobilized as,for example, on a solid support. It will be appreciated that the term“solid support” includes semi-solid supports as well. Once immobilized,the acceptor moiety is contacted with a mixture suspected to containglycosyltransferases, such as one comprising naturally-occurring cellhomogenate. Since an immobilized acceptor moiety will bind an enzymespecific for it, this system is then monitored for acceptor-boundenzyme.

[0056] Monitoring for acceptor-bound enzyme may be carried out asfollows. The cell homogenate is passed over the immobilized acceptormoiety. This may be achieved, for example, by passing the cellhomogenate over a column charged with immobilized acceptor moiety. Thecolumn is then washed and the amount of protein which passes through thecolumn charged with immobilized acceptor moiety is monitored. When nomore protein is detected, an aqueous salt solution eluant is passedthrough the column to elute the enzyme. The eluant obtained is thenassayed for the presence of glycosyltransferase(s). The assays which canbe used are noted above, i.e., the methods described by Furukawa et al,Roth et al and Benau et al.

[0057] If no binding of the enzyme to the acceptor moiety occurs (i.e.,the assay of the eluate fails to reveal the presence ofglycosyltransferase(s) therein), then it can be concluded that themixture did not contain an enzyme specific for the particular acceptor.Other mixtures of, for example, animal and/or plant cell homogenates arethen contacted with the acceptor moiety until enzyme binding isobserved.

[0058] When the acceptor moiety is bound by an enzyme, the species areseparated and further studied. In a preferred embodiment, the acceptorand the candidate enzyme are again contacted, this time in the presenceof a donor moiety which comprises the saccharide unit desired to betransferred to the exceptor moiety. If such contacting results in thetransfer of the saccharide unit to the acceptor, the enzyme is aglycosyltransferase useful in the practice of this invention.

[0059] It will be appreciated that once the glycosyltransferase isidentified, it can be sequenced and/or replicated by techniqueswell-known to those skilled in the art. For example, replication mightbe accomplished by recombinant techniques involving the isolation ofgenetic material coding for the glycosyltransferase and the preparationof an immortal cell line capable of producing the glycosyltransferase.Replication will likely prove desirable for commercial scale productionof saccharide compositions in accordance with this invention.

[0060] After the glycosyltransferase is identified, it is contacted withthe acceptor moiety and donor moiety under conditions sufficient toeffect transfer and covaiently bonding of the saccharide unit to theacceptor moiety. It will be appreciated that the conditions of, forexample, time, temperature, and pH appropriate and optimal for aparticular saccharide unit transfer can be determined by one of skill inthe art through routine experimentation. Certain co-reagents may alsoprove useful in effecting such transfer. For example, it is preferredthat the acceptor and donor moieties be contacted with theglycosyltransferase in the presence of divalent cations, especiallymanganese cations such as may be provided by MnCl₂.

[0061] In a preferred embodiment, the glycosyltransferase is immobilizedby attachment to a solid support and the acceptor and donor moieties tobe contacted therewith are added thereto. As discussed above, theglycosyltransferase used in accordance with the present invention isfrequently a mixture of glycosyltransferases containing at least oneglycosyltransferase possessing the desired activity, but purified singleglycosyltransferases may also be used in accordance with the presentinvention. In this preferred embodiment, either the mixture ofglycosyltransferases or the purified single glycosyltransferase may beimmobilized. Alternatively, the glycosyltransferase, donor and acceptorare each provided in solution and contacted as solutes.

[0062] A preferred procedure for immobilization ofglycosyltransferases—and of acceptor moieties, where necessary—is basedon the copolymerization in a neutral buffer of a water solubleprepolymer such as poly(acrylamide-co-N-acryloxysuccinimide (PAN), across-linking diamine such as triethylenetetramine, and theglycosyltransferase, as disclosed by Pollack et al., J. Am. Chem. Soc.(1980) 102:6324-36. The immobilization of the enzymes on PAN is usefulbecause small amounts of enzyme can be used, high yields of enzymeactivity are obtained, and the bond between enzyme and polymer isstable.

[0063] More preferred methods of immobilization include immobilizationof the glycosyltransferase amino groups onto solid support oxiranegroups (see, e.g., Chun et al, Enzyme Eng. (1980) 5:457-460) or ontocyanogen bromide activated “SEPHADEXII or “SEPHAROSE” (Axen et al,Nature (1967) 214:1302-1304).

[0064] In a preferred embodiment, the glycosyltransferase is immobilizedfrom a moderately purified composition containing theglycosyltransferase. Extremely pure enzyme preparations (i.e., withspecific activities in the range of 1 nMole transferred per μg proteinper minute of incubation) are less efficiently immobilized covalently tosolid supports, in that the percent derivatization is lower, compared to10 or 100 times less pure preparations.

[0065] It will be appreciated that impairment of the active sites of theglycosyltransferase due to immobilization should be avoided. Theinventor observed that contaminating enzyme activities tend to disappearduring the immobilization process as compared to the activity of theglycosyltransferase of interest which is specifically protected duringthe immobilization process. During the immobilization process theglycosyltransferase may be protected by the cation required by theenzyme, the nucleotide recognized by the enzyme, and the acceptorrecognized by the enzyme. For example, a galactosyl transferase may beprotected with Mn²⁺, N-acetylglucosamine and UDP during theimmobilization, regardless of which immobilization method is used. Inthis way, contaminating proteases are not protected in any way duringthe immobilization process.

[0066] Because only the desired glycosyltransferase is protected duringthe immobilization process, enzymes that interfere with the synthesis ofthe target saccharide composition tend to be lost. Examples ofinterfering enzymes are proteases, which would otherwise attack thedesired glycosyltransferase, and glycosidases, which would otherwiseattack the product saccharide.

[0067] As noted above, in accordance with the invention, a saccharidecomposition prepared by contacting an acceptor moiety with a donormoiety and a glycosyltransferase can, in turn, serve as an acceptormoiety for isolating further enzymes and as an acceptor moiety to whichsubsequent saccharide units may be transferred. The addition ofsaccharide units to saccharide compositions prepared by such contact ispreferred for the synthesis of carbohydrates and saccharide chainshaving greater than about three saccharide units.

[0068] For example, in preparing the trisaccharide N-acetylneuraminyl α2-3 galactosyl β 1-4 N-acetylglucosamine, the disaccharide galactosyl β3 1-4 N-acetylglucosamine is prepared according to this invention andthen employed as an acceptor moiety to which a subsequent unit is added.Those skilled in the art will appreciate that the saccharide unitsattached to the saccharide compositions of this invention can be thesame or different.

[0069] The saccharide compositions of this invention find use in anexceedingly wide variety of applications and may be used in the samemanner as saccharide compositions available from known sources. It ispreferred that the saccharide compositions be employed in therapeuticand preventative treatments for mammals, such as disclosed in U.S. Ser.No. 07/241,012.

[0070] The saccharide compositions of this invention are expected tofind use as blocking agents for cell surface receptors in the treatmentof numerous diseases of viral, bacterial, or fungal origins, such aspneumonia, candidiasis, urinary tract infections, periodontal disease,and diarrhea. For example, oligosaccharides prepared according to thisinvention may inhibit the attachment of pathogens such aspneumonia-causing bacteria to mammalian membrane molecules. Suchpathogens might be incubated with cellular glycoproteins and glycolipidsthat have been separated by chromatography or electrophoresis. Afterdetecting specific adherence patterns, the target compound could beanalyzed and inhibitory saccharide composition prepared. If either ofthe complimentary molecules functions through its saccharide component,then specific saccharide compositions should inhibit attachment.

[0071] The saccharide compositions which can be prepared in accordancewith the invention can be used in the following applications:

[0072] The present invention thus also provides pharmaceutical and othercompositions, such as foodstuff compositions, containing saccharidecompositions prepared in accordance with the present invention. In boththe pharmaceutical compositions and the foodstuff compositions providedby the invention, the saccharide composition of the invention may bepresent in an amount of from 10⁻³ μg ml⁻¹ to 100 mg ml⁻¹.

[0073] The concentration of the saccharide composition of the presentinvention in any given particular pharmaceutical composition orfoodstuff composition will vary in terms of the activity of thesaccharide being used. For pharmaceutical compositions the concentrationof saccharide present in the composition will depend on the in vitroactivity measured for any given compound. For foodstuff compositions,the concentration of the saccharide composition of the present inventionmay be determined measuring the activity of the compound being added.

[0074] For example, mother's milk contains the saccharide compositionset forth above where it is indicated as being useful both in infantformula and as an antibacterial for fighting urinary tract infections.As such, the present invention provides an improvement in commercialinfant formulas by permitting the addition to these commercial infantformulas the saccharide composition illustrated above. The particularsaccharide composition illustrated above may be present in thecommercial infant formula in an amount of 0.1 μg per ml to 1000 μg perml. It is present in mother's milk at ca. 10 μg per ml.

[0075] The pharmaceutical compositions should be pyrogen free.Pharmaceutical compositions in accordance to the present invention maybe prepared as is known in the art so as to be suitable for oral,intravenous, intramuscular, rectal, transdermal or nasal (e.g., nasalspray) administration. It may also be prepared for topicaladministration in the form of creams, ointments, suspensions, etc.

[0076] A few saccharides have been noted as being important both ascommodity chemicals in the food, textile, and petroleum industries, andas specialty chemicals, primarily in the medical field. To date, theabsence of an efficient process for preparing saccharide compositionshas made it impossible to obtain commercial compositions containing, asan active ingredient, a saccharide composition.

[0077] The present invention makes such saccharide compositions readilyavailable in large quantity for the first time. With the method of thepresent invention, saccharide compositions heretofore available only inminiscule quantities, and saccharide compositions heretoforeunavailable, are readily made in gram and kilogram quantities. Thepurity of the saccharide compositions provided in accordance to thepresent invention exceeds 95 wt. %. In some applications requiring ahigh level of purity, the method of the present invention can be used toobtain saccharide compositions containing purity levels of from 98 wt. %to essentially 100 wt. %.

[0078] The present invention thus now provides for the first timepharmaceutical compositions and other compositions containing saccharidecompositions present invention present in an effective amount. Thepresent invention provides compositions containing the saccharidecompositions obtained in accordance with the present invention presentin the amount of at least 100 mg, preferably at least 500 mg, and up to95 wt. % of the composition.

[0079] In another embodiment, the present invention provides anapparatus suitable for use in accordance with the present invention forthe glycosyltransferase catalyzed synthesis of a saccharide composition.Illustrative configurations for such apparatus are provided in FIGS. 1,2 and 3.

[0080] In a very basic embodiment the apparatus of the present inventioncontains one reaction chamber in which all of the glycosyltransferases,all the preselected saccharide units and the initial acceptor moiety arecombined. Due to the specificity of the glycosyltransferases, thismixture, given sufficient time, will produce the saccharide compositionof the present invention.

[0081]FIGS. 1, 2 and 3 illustrate more efficiently designed apparatuseswhich may be used in accordance with the present invention. Theapparatuses illustrated in the figures, comprise, as their basicelements, a reactor equipped with an inlet and an outlet. The reactor issuitable for carrying out the sequential covalent bonding of a pluralityof preselected saccharide units onto an acceptor moiety, catalyzed by aplurality of corresponding glycosyltransferases specific to eachcovalent bonding. It contains at least three, preferably four, and evenmore preferably a number greater than four, such as five, six, seven, ormore, different, glycoltransferases which are preferably immobilized.

[0082] The inlet means is suitable for introducing the acceptor moietyand the plurality of preselected saccharide units into the reactor suchthat the saccharide composition is synthesized. Preferably, the inletmeans is suitable for also introducing into the reactor theglycosyltransferases which are themselves preferably immobilized. Theoutlet means permits discharging the saccharide composition from thereactor.

[0083]FIG. 1 illustrates a column-type reactor charged with a solidsupport matrix. The various glycosyltransferases (enzymes 1, 2, 3) usedin the process may be either randomly distributed throughout the solidsupport matrix or they may be arranged in zones as illustrated inFIG. 1. The initial acceptor moiety (shown as A in the figures) and thepreselected saccharide units (shown as B, C and D in the figures) arecharged into the reactor via the inlet means and passed through thesolid support matrix whereupon the saccharide composition is produceddue to the action of the specific glycosyltransferases and recovered viathe outlet means as molecule A-B-C-D.

[0084] In the embodiment illustrated in FIG. 2, the initial acceptormoiety and the preselected saccharide unit to be attached to the initialacceptor moiety are charged at the top of the solid support matrix, withthe glycosyltransferases specific to the addition of each preselectedsaccharide units being arranged in corresponding zones along thedirection of flow of the reaction mixture. The various preselectedsaccharide units are then individually added at correspondinglyappropriate locations along the flow of the reaction mixture as shown inthe figure.

[0085] In another preferred embodiment, illustrated in FIG. 3, thereactor comprises a plurality of (n) reaction zones serially connectedso as to be in sequential fluid communication with each other where (n)roughly corresponds to not more than the number of saccharide unitsbeing attached. Each reaction zone contains at least oneglycosyltransferase specific to catalyze the bonding of a particularpreselected saccharide unit onto the intermediate product formed in thepreceding reaction zone.

[0086] In accordance with this embodiment the initial acceptor moiety(A) and the first preselected saccharide unit (B) to be attached to theacceptor moiety are passed through the first reaction zone whichcomprises a glycosyltransferase specific to catalyze the bonding of thefirst preselected saccharide unit onto the initial acceptor moiety thusproducing a first intermediate product. This first intermediate productis then transferred to the second reaction zone (n=1) where it iscombined with the second preselected saccharide unit (X_(n)) and theglycosyltransferase (E_(1+n)) specific to catalyze the bonding of thesecond preselected saccharide unit with the first intermediate productformed. This process is repeated in a corresponding number of reactionzones until the target saccharide composition provided by the inventionand illustrated as A-B-(X)-_(n)Z, wherein each X moiety is independentlyselected and n is an integer of from 1 to 500 or more, is obtained.

[0087] In another preferred embodiment, also illustrated in FIG. 3,means for purifying 4 each intermediate product formed from the reactionmixture emanating from any given reaction zone are situated in fluidcommunication and between each of the reaction zones. The means forpurifying, which may comprise, e.g., an ion exchange resin, removecontaminants in the reaction mixtures which inhibit the efficiency ofthe bonding of the next preselected saccharide unit onto theintermediate product formed.

[0088] Additional objects, advantages, and novel features of thisinvention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are not intended tobe limiting.

EXAMPLE 1 Preparation of the Trisaccharide N-Acetylneuraminyl α 2-3Galactosyl β 1-4 N-Acetylglucosamine:

[0089] To each of five test tubes was added 10 μl of pH 7.4 potassiumphosphate buffer, 10 μl of 50 MM MnCl₂, 17,000 CPM of cytidinemonophosphate-[¹⁴C]-N-acetylneuraminic acid, 25 μl ofgalactosyltransferase, and 25 μl of N-acetylneuraminyltransferase. Theglycosyltransferases were purified from bovine colostrum by SephadexG-100 gel chromatography.

[0090] To test tube 1 was also added 10 μl of 40 mm uridine diphosphategalactose and 10 μl of 40 mM N-acetylglucosamine. Test tube 1 wasincubated in ice for one hour.

[0091] To test tube 2 was also added 10 μl of 40 mM uridine diphosphategalactose. Test tube 2 was incubated at 37° C. for one hour.

[0092] To test tube 3 was also added 10 μl of 40 mM N-acetyllactosamine.Test tube 3 was incubated at 37° C. for one hour.

[0093] To test tubes 4 and 5 were also added 10 μl of 40 mM uridinediphosphate galactose and 10 μl of 40 mM N-acetylglucosamine. Test tubes4 and 5 were incubated at 37° C. for one hour.

[0094] After incubation, the contents of the test tubes were eachsubjected to high voltage electrophoresis on paper saturated with sodiumtetraborate. Isotopically labeled trisaccharide product was identifiedby its mobility, as demonstrated by the product formed in test tube 3.Test Tube Trisaccharide (cpm) 1 0 2 0 3 3375 4 670 5 954

[0095] As can be seen, the presence of suitable acceptor moieties, donormoieties, and glycosyltransferases in test tubes 4 and 5 yielded theexpected trisaccharide product from monosaccharide starting materials.Typically, the sialic acid N-acetylneuraminate presents special problemsfor synthetic organic chemists seeking to incorporate it into saccharidecompositions, due to the acid lability of its glycosidic bond.Synthesizing a trisaccharide from cytidine monophosphateN-acetylineuraminic acid enzymatically eliminates the synthetic problemsassociated with removing protecting groups under strong acidiccondition.

[0096] It is believed that an acceptor moiety (N-acetylglucosamine)initially contacts a donor moiety (uridine diphosphate galactose) and aglycosyltransferase (galactosyltransferase) to produce a saccharidecomposition (galactosyl β 1-4 N-acetylglucosamine), which then acts asan acceptor moiety upon contacting a second donor moiety (cytidinemonophosphate N-acetylneuraminic acid) and a second glycosyltransferase(N-acetylneuraminyltransferase).

[0097] The synthesis of the trisaccharide product in test tubes 4 and 5from monosaccharide starting materials is confirmed by comparison withthe product of test tube 3, in which the trisaccharide is formed bycontacting a disaccharide acceptor moiety (N-acetyllactosamine) withcytidine monophosphate N-acetylneuraminic acid andN-acetylneuraminyltransferase.

[0098] The absence of trisaccharide in test tube 2 illustrates that asuitable acceptor moiety is necessary for trisaccharide formation. Theabsence of trisaccharide in test tube 1 indicates that the synthesis ofthe trisaccharide is, indeed, dependent upon the action of any enzyme(the glycosyltransferase) that is inactive at low temperatures.

[0099] It is expected that the oligosaccharides N-acetylgalactosaminyl α1-3 (fucosyl α 1-2) galactosyl β 1-4 N-acetylglucosaminyl β 1-3galactose (a target for diarrhea-causing bacteria) andN-acetylgalactosaminyl β 1-4 galactosyl β 1-4 glucose (a target forpneumonia-causing bacteria) can likewise be prepared by the processes ofthe present invention.

EXAMPLE 2 Tetrasaccharide Biosynthesis protocol EnzymesN-acetylglucosaminyltransferase

[0100] Human colostrum is centrifuged for one hour at 70,000×G. A 25%saturated ammonium sulfate cut yields a supernatant that is dialyzed toremove the ammonium sulfate. The retentate is applied to a SephadexG-200 column (2.5×83 cm). The protein profile is determinedspectrophotometrically at 280 nm, and a radioactive assay is performedto locate the fractions with transferase activity. The fractionscontaining the single enzyme peak are pooled and concentrated 10-fold byAmicon filtration. The pooled enzyme preparation is again assayed, andthe protein concentration is determined using a BioRad assay. Thespecific activity of the preparation is 5.3 pMoles per μg protein-min.

Galactosyltransferase

[0101] Human colostrum is centrifuged at 8700×G for 15 minutes. Thesupernatant is poured through cheesecloth and 10 ml is applied to aSephadex G-100 column (2.5×90 cm). The protein profile is determinedspectrophotometrically at 280 nm, and a radioactive assay is performedto locate the fractions with enzyme activity. The fractions with thehighest activity are pooled and concentrated 10-fold by Amiconfiltration. The pooled enzyme preparation is again assayed, and theprotein concentration is determined as above. The specific activity ofthe preparation is 15.4 pMoles per μg protein-min.

Enzyme immobilization N-acetylglucosaminyltransferase

[0102] 300 mgs of Eupergit beads (1.2 ml) are washed three times withdeionized water, and then three times with aseptic Hepes-buffered water.One ml of the enzyme preparation is combined aseptically with the beadsalong with UDP, lactose, MnCl₂, (final concentrations: 10, 25, and 10mM, respectively) and a drop of chloroform in a Hepes-buffered solution.The beads are gently agitated at 4° C. for 2½ days. Aliquots are takenand assayed periodically. To stop the derivatization, the beads arewashed three times with an aseptic buffer, and stored in buffer, in thecold, with UDP, lactose, MnCl₂, and chloroform.

Galactosyltransferase

[0103] 3.75 grams of beads are washed three times with deionized water,and then three times with aseptic Hepes-buffered water. The beads areadded to 3-mls of the enzyme preparation (in both cases, optimumderivatization occurs at about 1 mg protein per 200 mgs beads) alongwith UDP, GlcNAc, MnCl₂, (final concentrations are all 10 mM) and a dropof chloroform in a Hepes-buffered solution. Derivatization and storageare as described above, except that the GlcNac is used with thegalactosyltransferase in place of lactose, which is the acceptor for theN-acetylglucosaminyltransferase.

Tetrasaccharide production

[0104] Derivatized N-acetylglucosaminyltransferase (0.5 ml beads) isincubated under constant stirring with lactose (25 mM), UDPGlcNAc (80μM), and MnCl₂ (10 mM) for 21 hours. This incubation is carried out induplicate-the supernatant of one incubation is used to measure theamount of trisaccharide produced (14 μg), and the supernatant from theother incubation is added to 0.5 ml beads derivatized with thegalactosyltransferase. The galactosyltransferase incubation contains,therefore, 14 μg of trisaccharide, 25 μM UDPgal, and 10 mM MnCl₂. After24 hours at room temperature, the second enzyme preparation producesabout 1.6 μg of tetrasaccharide. After 31 hours, 2.2 μg oftetrasaccharide were produced.

EXAMPLE 3

[0105] The following schemes will be used for synthesizing three,relatively complex oligosaccharides: the A- and B-type milkoligosaccharides (I and II), and gum tragacanth (III), a plantoligosaccharide used by the ton as a food additive.

galNACα1,→(fucα1,2→)galβ1,3→(fucα1,4→) GlcNAcβ1,3→galβ1,4→glc  (I)

[0106] First, the hexanolamine glycoside (glc-O—(CH₂)₆—NH₂) of glucosethat will be affixed to CNBr-activated supports, e.g., Sepharose, viathe amino group of the hexanolamine will be synthesized. Then theglucose-recognizing galactosyltransferase will be purified from humanmilk or colostrum using this affinity ligand. The enzyme, once partlypurified, will be used to galactosylate glucose, making lactose.

[0107] Alternatively, the hexanolamine glycoside of lactose, which is aninexpensive and readily available disaccharide, will be synthesized. Thelactose so produced will be attached to Sepharose and used as anaffinity ligand to purify in part the N-acetylglucosaminyltransferasefrom human colostrum, or from human plasma.

[0108] Next, this second transferase will be used to addN-acetylglucosamine to lactose, making the trisaccharide, which willagain be attached to Sepharose. This bound trisaccharide will be used toobtain the β1,3 galactosyltransferase (from porcine submaxillary gland),which will, in turn, yield the substrate for purifying the nextenzyme—the α1,4 fucosyltransferase (from porcine liver). The α1,2fucosyltransferase (from porcine submaxillary gland), and, finally, theα1,3 N-acetylgalactosaminyltransferase (from porcine submaxillaryglands) that terminates the synthesis of the A-type milk oligosaccharidewill be affinity purified in this step-wise fashion. Each transferase soobtained will be immobilized to a solid matrix by any of several means,and the matrices will be poured in column configurations.

[0109] The enzyme-containing columns will be used sequentially, in thesame order that the smaller amounts of derivatized substrates weresynthesized, to synthesize large amounts each soluble oligosaccharide.

[0110] The order of attachment of the sugars is critical. The proximalfucose (that attached α1,4 to glcNAc) must be attached to the completedcore tetrasaccharide before the addition of the second fucose (thatattached α1,2 to the galactose. Finally, the terminal galNAc (α1,3) isadded to complete the seven-sugar oligosaccharide. This order isrequired by the specificities of the glycosyltransferases.

galα1,3→(fucα1,2→)galβ1,3→(fucα1,4→) GlcNAcβ1,3→galβ1,4→glc  (II)

[0111] Having synthesized I, II will be synthesizes in precisely thesame fashion, except that the hexasaccharide will be used, first, topurify an α13 galactosyltransferase that will be derivitized withprotective groups for a galactosyl-, and not anN-acetylgalactosaminyltransferase. This enzyme will then be used tosynthesize the B-type oligosaccharide.

[. . . (fucα1,3→xylβ1,3→)galAα1,4→(gal,β1,4→xylβ1,3→)galA . . . ]  (III)

[0112] To isolate the enzyme that synthesizes the α1,4 galacturonic acidbackbone of gum tragacanth, which currently is available only from thebark of a tree species indigenous to the Middle East, hexagalacturonanswill be prepared from pectin, a common constituent of citrus rinds, andused as an affinity ligand.

[0113] The same affinity ligand can next be used to isolate from treetissues the xylosyltransferase that synthesizes the proximal β1,3xylosides. The xylosylated galacturonans, once derivatized, will be usedto isolate both the fucosyl- and galactosyltransferases that,respectively, fucosylate and galactosylate the xylosylated galacturonan.In the case of this oligosaccharide, the degree of xylosylation,fucosylation, and galactosylation will be controlled empirically by thenumber of passes of the compounds through the appropriateenzyme-containing columns. The number of repeat units produced willdepend on the number of galacturonic acid residues used initially; thisnumber will vary in length from four to twenty monosaccharide units.

[0114] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method for the glycosyltransferase-catalyzedpreparation of a saccharide composition by serially bonding preselectedsaccharide units to an acceptor moiety, comprising the steps of: (i)preparing a glycosyltransferase capable of transferring a preselectedsaccharide unit to an acceptor moiety by contacting said acceptor moietywith a mixture suspected to contain a plurality of glycosyltransferasesunder conditions effective to bind said acceptor moiety and saidglycosyltransferase, and thereby isolating said glycosyltransferase,wherein said acceptor moiety is one member selected from the groupconsisting of proteins, glycoproteins, lipids, glycolipids, andcarbohydrates; (ii) providing conditions and co-reagents sufficient toeffect bonding of said preselected saccharide unit to said acceptormoiety catalyzed by said glycosyltransferase thereby obtaining aproduct; and (iii) performing steps (i) and (ii) a plurality of timessuch that the product obtained in step (ii) of a given iteration is usedas the acceptor moiety in step (i) of the following iteration until saidsaccharide composition is obtained.
 2. The method of claim 1 whereinsaid carbohydrate is one member selected from the group consisting ofmonosaccharides, disaccharides, oligosaccharides, and polysaccharides.3. The method of claim 1 wherein the glycosyltransferase used in step(ii) is immobilized to a solid support.
 4. The method of claim 3 whereinsaid glycosyltransferase attached to a solid support is obtained byprotecting the active site of said glycosyltransferase during theimmobilization process.
 5. The method of claim 1 wherein saidco-reagents comprise manganese cations.
 6. The method of claim 1 whereinsaid saccharide used in step (ii) is a saccharide nucleotide.
 7. Themethod of claim 5 wherein said nucleotide is one member selected fromthe group consisting of uridine, guanosine and cytidine phosphates. 8.The method of claim 1 wherein said acceptor moiety used in said firstiteration is one member selected from the group consisting of proteins,glycoproteins, lipids, and glycolipids.
 9. The method of claim 1 whereinsaid acceptor moiety used in said first iteration is one member selectedfrom the group consisting of monosaccharides, disaccharides,oligosaccharides, and polysaccharides.
 10. The method of claim 1 whereinsaid acceptor moiety used in said first iteration isN-acetylglucosamine.
 11. The method of claim 1 wherein said acceptormoiety used in said first iteration is N-acetylglucosamine, saidglycosyltransferase used in said first iteration isgalactosyltransferase, and said glycosyltransferase used in said seconditeration is N-acetylneuraminyltransferase.
 12. The method of claim 1wherein said acceptor moiety used in said second iteration is galactosylβ 1-4 N-acetylglucosamine.
 13. The method of claim 1 wherein at leastone of the donor moieties used is cytidine monophosphateN-acetylneuraminic acid.
 14. The method of claim 1 wherein said mixturesuspected to contain a plurality of glycosyltransferases is a cellhomogenate.
 15. The method of claim 1 wherein said saccharidecomposition is a compound of one of the following formulae:


16. A pharmaceutical composition comprising, in association withpharmaceutically acceptable excipient or carrier, a saccharidecomposition other than heparin prepared by a method forglycosyltransferase-catalyzed serial bonding of preselected saccharideunits to an acceptor moiety comprising the steps of: (i) preparing aglycosyltransferase capable of transferring a preselected saccharideunit to an acceptor moiety by contacting said acceptor moiety with amixture suspected to contain said glycosyltransferase under conditionseffective to bind said acceptor moiety and said glycosyltransferase,thereby isolating said glycosyltransferase, wherein said acceptor moietyis one member selected from the group consisting of proteins,glycoproteins, lipids, glycolipids, and carbohydrates; (ii) providingconditions and co-reagents sufficient to effect bonding of saidpreselected saccharide unit to said acceptor moiety catalyzed by saidglycosyltransferase thereby obtaining a product; and (iii) performingsteps (i) and (ii) a plurality of times such that the product obtainedin step (ii) of a given iteration is used as the acceptor moiety of step(i) of the following iteration.
 17. The pharmaceutical composition ofclaim 16 comprising at least 100 mg of said saccharide composition. 18.The pharmaceutical composition of claim 16 comprising at least 1 gram ofsaid saccharide composition.
 19. The pharmaceutical composition of claim16 wherein said acceptor moiety used in said first iteration is aprotein.
 20. The pharmaceutical composition of claim 16 wherein saidacceptor moiety used in said first iteration is a glycoprotein.
 21. Thepharmaceutical composition of claim 16 wherein said acceptor moiety usedin said first iteration is a lipid.
 22. The pharmaceutical compositionof claim 16 wherein said acceptor moiety used in said first iteration isa glycolipid.
 23. The pharmaceutical composition of claim 16 whereinsaid acceptor moiety used in said first iteration is a carbohydrate. 24.The pharmaceutical composition of claim 16 wherein said acceptor moietyused in said first iteration is a monosaccharide.
 25. The pharmaceuticalcomposition of claim 16 wherein said acceptor moiety used in said firstiteration is a disaccharide.
 26. The pharmaceutical composition of claim16 wherein said acceptor moiety used in said first iteration is anoligosaccharide.
 27. The pharmaceutical composition of claim 16 whereinsaid acceptor moiety used in said first iteration is a polysaccharide.28. A pharmaceutical composition suitable for use in the therapy ortreatment of pneumonia comprising an effective amount of a compound ofthe formula:

in association with a pharmaceutically acceptable carrier or excipient.29. A pharmaceutical composition suitable for use in the therapy ortreatment of periodontal disease comprising an effective amount of acompound of the formula:

in association with a pharmaceutically acceptable carrier or excipient.30. A pharmaceutical composition suitable for use in the therapy ortreatment of solid tumors comprising an effective amount of a compoundof the formula:

in association with a pharmaceutically acceptable carrier or excipient.31. A pharmaceutically composition suitable for use as a contraceptivecomprising an effective amount of a compound comprising the formula:

in association with a pharmaceutically acceptable carrier or excipient.32. A foodstuff comprising a saccharide composition other than gumtragacanth or carrageenan prepared by a method forglycosyltransferase-catalyzed serial bonding of preselected saccharideunits to an acceptor moiety comprising the steps of: (i) preparing aglycosyltransferase capable of transferring a preselected saccharideunit to an acceptor moiety by contacting said acceptor moiety with amixture suspected to contain a plurality of glycosyltransferases underconditions effective to bind said acceptor moiety and saidglycosyltransferase, and thereby isolating said glycosyltransferase,wherein said acceptor moiety is one member selected from the groupconsisting of proteins, glycoproteins, lipids, glycolipids, andcarbohydrates; (ii) providing conditions and co-reagents sufficient toeffect bonding of said preselected saccharide unit to said acceptormoiety catalyzed by said glycosyltransferase thereby obtaining aproduct; and (iii) performing steps (i) and (ii) a plurality of timessuch that the product obtained in step (ii) of a given iteration is usedas the acceptor moiety in step (i) of the following iteration.
 33. Ininfant foodstuff, the improvement comprising a compound of the formula:

present in an amount of from about 0.1 μg ml⁻¹ to about 1000 μg ml⁻¹.34. An apparatus for the glycosyltransferase-catalyzed synthesis of asaccharide composition, said apparatus comprising: a reactor; at leastfour different glycosyltransferases in said reactor; inlet means forintroducing an acceptor moiety and a plurality of preselected saccharideunits into said reactor such that said saccharide composition issynthesized; and outlet means for discharging said saccharidecomposition from said reactor; wherein said acceptor moiety is onemember selected from the group consisting of proteins, glycoproteins,lipids, glycolipids, and carbohydrates.
 35. The apparatus of claim 34wherein said reactor comprises a plurality of reaction zones seriallyconnected so as to be in sequential fluid communication with each other,wherein each reaction zone comprises a glycosyltransferase specific tocatalyze bonding of a preselected saccharide unit onto the intermediateproduct formed in the preceding reaction zone.
 36. The apparatus ofclaim 34 wherein means for purifying the intermediate products formed ineach of said reaction zones from the rest of the reaction mixtureproduced therein is situated in fluid communication and between each ofsaid reaction zones.
 37. The apparatus of claim 34 wherein saidglycosyltransferases are immobilized onto a solid support.
 38. Theapparatus of claim 37 wherein the active sites of saidglycosyltransferases immobilized onto said solid support are protectedduring the immobilization process.